Compounds from antrodia cinnamomea and use thereof

ABSTRACT

The present invention relates to compounds from  Antrodia cinnamomea . The present invention also relates to a composition and a method for treating or prophylaxis of hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infection.

FIELD OF THE INVENTION

The present invention relates to compounds from Antrodia cinnamomea. Thepresent invention also relates to a composition and a method fortreating or prophylaxis of hepatitis C virus (HCV) or humanimmunodeficiency virus (HIV) infection.

BACKGROUND OF THE INVENTION

It is estimated that approximate 3% of the world's population isinfected with hepatitis C virus (HCV). In developed countries, chronichepatitis C is the leading cause for cirrhosis, hepatocellularcarcinoma, and liver transplantation. The protease of hepatitis C virusis required for the cleavage of viral nonstructural polyprotein to formthe mature virus and represents one of the attractive therapeutictargets for developing antiviral agents against HCV (Liu et al., 2004;Hepatitis C NS3 protease inhibition by peptidyl-a-ketoamide inhibitors:kinetic mechanism and structure. Arch Biochem Biophys 421: 207-216;Kakiuchi et al., 1999 A high throughput assay of the hepatitis C virusnonstructural protein 3 serine proteinase. J Virol 80: 77-84).

Human immunodeficiency virus (HIV) is the virus known to cause acquiredimmunodeficiency syndrome (AIDS) in humans and AIDS presents specialproblems to the medical community which the present invention addresses.Without treatment, immunodeficiency viral infection is highly lethal.Indeed, AIDS is the leading cause of human death. In certain parts ofthe world, such as sub-Saharan Africa, at least 10% of all adults arebelieved to be infected with HIV, with the prevalence in many capitalcities believed to be 35% or more. In the United States, an estimated800,000 to 900,000 people are currently infected with HIV, withapproximately 40,000 new infections occurring each year. Of the morethan 700,000 individuals in the United States who were infected with HIVas of December 2000, 58% have died.

The use of herbal therapy and folk medicines has been known forthousands of years in China. In fact, records on the use of herbs dateback to biblical times. However, only recently have scientists begunexploring the possible role for herbs in treatment of viral infections.For example, extracts from the root of the Ecballium elaterium have beenused to treat HCV and HBV (EP 0793964 and U.S. Pat. No. 5,648,089).While research in the field of herbal medicines has increased, muchremains to be learned about the effectiveness of such herbal remedies.

The fruiting body of Antrodia cinnamomea T. T. Chang & W. N. Chou (ataxonomic synonym of Antrodia camphorate, referring to Wu et al., 1997,Antrodia camphorata (“niu-chang-chih”), new combination of a medicinalfungus in Taiwan. Bot. Bull. Acad. Sin. 38: 273-275) is a highly valuedfolk medicine in Taiwan. It is used as an antidote and for diarrhea,abdominal pain, hypertension, itchy skin, and liver cancer. Somebioactive constituents from the fruiting body of Antrodia cinnamomeahave been isolated and characterized as a series of polysaccharides,steroids, triterpenoids, and sesquiterpene lactone (Lin et al., 2007,Factors affecting mycelial biomass and exopolysaccharide production insubmerged cultivation of Antrodia cinnamomea using complex media.Bioresource Technology 98: 2511-2517). In previous studies, five newmaleic and succinic acid derivatives (Compound 1-5) are isolated fromthe mycelium of Antrodia cinnamomea (Nakamura et al., 2004, Five newmaleic and succinic acid derivatives from the mycelium of Antrodiacomphorata and their cytotoxic effects on LLC tumor cell line. J NatProd 67: 46-48).

U.S. Pat. No. 7,109,232 discloses Compounds 1-5 from Antrodia cinnamomeaand their uses such as hepatoprotection, anti-inflammation or anti-tumoractivity and preparation. PCT Publication No. WO 2009/094807 A1discloses Compounds 1-10 from Antrodia cinnamomea and their use fortreating or prophylaxis of hepatitis C virus infection.

Traditionally, the fruit body of Antrodia cinnamomea has been used forliver cancer (Lin E S, Chen Y H. 2007. Factors affecting mycelialbiomass and exopolysaccharide production in submerged cultivation ofAntrodia cinnamomea using complex media. Bioresource Technology 98:2511-2517). Polysaccharides of Antrodia cinnamomea has been show to havehepatoprotective effect (Han et al., 2006b, Protective effects of aneutral polysaccharide isolated from the mycelium of Antrodia cinnamomeaon Propionibacterium acnes and lipopolysaccharide induced hepatic injuryin mice. Chem Pharm Bull 54: 496-500) and anti-hepatitis B virusactivity (Lee et. al., 2002, Antrodia camphorate polysaccharides exhibitanti-hepatitis B virus effects. FEMS Microbiol Lett 209:63-67). Of themaleic and succinic acid derivatives, Compound 3 showed protectiveactivity in Propionicbacterium acnes and lipopolysaccharide treated mice(Nakamura N, Five new maleic and succinic acid derivatives from themycelium of Antrodia comphorata and their cytotoxic effects on LLC tumorcell line. J Nat Prod 67: 46-48). Quantitative analysis showed thatCompound 3 was the most abundant compound of this chemical type in themycelium with a content of ca. 5% of the dry weight of mycelia (Han etal., 2006a, Protective effects of a neutral polysaccharide isolated fromthe mycelium of Antrodia cinnamomea on Propionibacterium acnes andlipopolysaccharide induced hepatic injury in mice. Chem Pharm Bull 54:496-500).

During the investigation for anti HCV agents from natural source,compounds isolated from Antrodia cinnamomea showed variable activities(PCT publication No. WO 2009/094807). The highest was compound 1 (0.9μg/mL), compound 3 (2.9 μg/mL). Compound 2 shows low activity, althoughit is sharing the main nucleus with other compounds. Therefore a seriesof derivatives was carried out, in this study, for Compound 2 targetingto improve its activity as anti HCV protease agent. Also, a new isolatedcompound from Antrodia cinnamomea was investigated its activity as antiHCV or HIV-1 protease agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ingredient isolated from the EtOH extract of themycelium of Antrodia cinnamomea.

FIG. 2 shows the HPLC chromatogram of Fr. 12.

FIG. 3 shows MS spectrum of a compound of formula (XIII).

FIG. 4 shows 2D NMR correlations for a compound of formula (XIII). Heavyline: ¹H-¹H COSY; One-way arrow: HMBC related; Two-way arrow: NOErelated. The curve line means that the compound of formula (XIII) is aracemic mixture of (4S)- and(4R)-[4-(3-methyl-2-butenyloxy)phenyl]derivatives with a 1:1 ratio. Thiswas confirmed by the optical rotation value [α]_(D)=0.

FIG. 5 shows the chromatogram of a compound of formula (XIII) with achiral column. Column: SHISEIDO FINE CHEMICALS Chiral CD-Ph PackedColumn (4.6 i.d.×250 mm) Temperature: 30° C.; shift layer: CH₃CN-0.1%Acetic acid=20:80→100:0; flow speed: 0.5 mL/min, detection: ESI-MS(+).

FIG. 6 shows MS² spectra for both peaks.

FIG. 7 shows principles of NS3/4A HCV protease inhibition assay.Fluorescence was generated when 5-FAMsp was cleaved. The fluorescenceintensity was detected.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide novelcompounds from the mycelium of Antrodia cinnamomea.

Another object of the present invention is to provide a pharmaceuticalcomposition for treating or prophylaxis of hepatitis C virus or humanimmunodeficiency virus infection, which comprises a compound of thepresent invention in an amount effective to attenuate infectivity ofsaid virus, and a pharmaceutically acceptable carrier.

Further object of the present invention is to provide a method fortreating or prophylaxis hepatitis C virus or human immunodeficiencyvirus infection which comprises administering to a subject in needthereof an effective amount of a composition comprising the compoundsfrom the mycelium of Antrodia cinnamomea.

Additional embodiments, features, and advantages of the invention willbe apparent from the following detailed description and through practiceof the invention.

DETAILED DESCRIPTION OF THE INVENTION

For the sake of brevity, the disclosures of the publications, includingpatents, cited in this specification are herein incorporated byreference.

The terms “including”, “containing” and “comprising” are used herein intheir open, non-limiting sense.

Accordingly, the present invention also provides a compound of formula(I)

where R₁ is

or H;

R₂ is selected from the groups as follows:

R₃ is C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, benzyl, or4-(3-methyl-2-butenyloxy)phenyl,

denotes the place of connection, or pharmaceutically acceptable salts orprodrugs thereof.

Preferably, R₁ is

R₂ is selected from the groups as follows:

R₃ is isobutyl,

denotes the place of connection.

More preferably, R₁ is H; R₂ is selected from the groups as follows:

R₃ is isobutyl,

denotes the place of connection.

The present invention also provides a compound selected from compoundsof formula (XIV), pharmaceutically acceptable salts of compounds offormula (XIV), and pharmaceutically acceptable prodrugs of compounds offormula (XIV),

wherein R is

denotes the place of connection.

Preferably, R is

More preferably, R is

The present invention also provides a compound having formula (II),

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl, or pharmaceutically acceptable saltsor prodrugs thereof.

Preferably, the compound is the compound of formula (XIII)

Certain compounds exist in one or more particular geometric, optical,enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric,conformational, or anomeric forms, including but not limited to, (+) and(−) forms; cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms;endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- andl-forms; keto-, enol-, and enolate-forms; syn- and anti-forms;synclinal- and anticlinal-forms; alpha.- and beta.-forms; axial andequatorial forms; boat-, chair-, twist-, envelope- and halfchair-forms;and combinations thereof, hereinafter collectively referred to as“isomers” (or “isomeric forms”).

If the compound is in crystalline form, it may exist in a number ofdifferent polymorphic forms.

Unless otherwise specified, the compounds of the present inventioninclude all such isomeric forms, including (wholly or partially) racemicand other mixtures thereof. Methods for the preparation (e.g. asymmetricsynthesis) and separation (e.g. fractional crystallization andchromatographic means) of such isomeric forms are either known in theart or are readily obtained by adapting the methods taught herein, orknown methods, in a known manner.

Unless otherwise specified, the compounds of the present invention alsoincludes ionic, salt, solvate, and protected forms of thereof. It may beconvenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

The pharmaceutically acceptable salts of the compounds are preparedfollowing procedures which are familiar to those skilled in the art.

The present invention also provides a pharmaceutical composition fortreating or prophylaxis of hepatitis C virus (HCV) infection whichcomprises an effective amount of an active agent selected from

(1) a compound selected from compounds shown in Table 9 or Table 10; or(2) a compound having the formula (II)

wherein R₁ is C₁₋₁₀alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; and a pharmaceutically acceptablecarrier.

Preferably, the compound has formula (XIV)

, wherein R is

denotes the place of connection.

More preferably, R is

Preferably, the subject is a mammal; more preferably, the subject is ahuman.

In another preferred embodiment, the compound is a compound of formula(XIII)

The present invention also provides a pharmaceutical composition fortreating or prophylaxis of human immunodeficiency virus (HIV) infectionwhich comprises an effective amount of an active agent selected from

(1) a compound having the formula (II)

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; or(2) a compound having the formula (II)

wherein

denotes a single or double bond;

X is N or O;

R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy;

R₂ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl; and

R₃ is absent, H or hydroxy; provided that if X is O, R₃ is absent;

and a pharmaceutically acceptable carrier.

Preferably, the compound is compound of formula (XIII)

In another preferred embodiment, the compound is

-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione,-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione,-   3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione,-   (3R*,4S*)-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione,    or-   (3R*,4R*)-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-pyrrolidine-2,5-dione.

Preferably, the subject is a mammal; more preferably, the subject is ahuman.

The composition may be prepared in various forms for administration,including tablets, caplets, pills or dragees, or can be filled insuitable containers, such as capsules, or, in the case of suspensions,filled into bottles. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the anti-viralcompounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. In thepharmaceutical compositions of the invention, the active agent may bepresent in an amount of at least 0.5% and not more than 90% by weightbased on the total weight of the composition, including carrier mediumand/or auxiliary agent(s). Preferably, the proportion of active agentvaries between 5%-50% by weight of the composition. Pharmaceuticalorganic or inorganic solid or liquid carrier media suitable for enteralor parenteral administration can be used to make up the composition.Gelatine, lactose, starch, magnesium, stearate, talc, vegetable andanimal fats and oils, gum, polyalkylene glycol, or other knownexcipients or diluents for medicaments may all be suitable as carriermedia.

The present invention further provides a method for treating orprophylaxis of hepatitis C virus (HCV) infection which comprisesadministering to a subject in need thereof an effective amount of anactive agent selected from

-   -   (1) a compound selected from compounds shown in Table 9 or Table        10;    -   (2) a compound having the formula (II)

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; and

R₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl.

In another preferred embodiment, the compound is a compound of formula(XIII)

-   -   (3) a compound selected from compounds of formula (XIV),        pharmaceutically acceptable salts of compounds of formula (XIV),        and pharmaceutically acceptable prodrugs of compounds of formula        (XIV)

wherein R is

denotes the place of connection; or

Preferably, R is

More preferably, R is

The present invention yet further provides a method for treating orprophylaxis of human immunodeficiency virus (HIV) infection whichcomprises administering to a subject in need thereof an effective amountof an active agent selected from

-   -   (1) a compound having the formula (II)

-   -    wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀        alkynyloxy; and        -   R₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; or    -   (2) a compound having the formula (III)

-   -   -   wherein        -   denotes a single or double bond;        -   X is N or O;        -   R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy;        -   R₂ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl; and        -   R₃ is absent, H or hydroxy;        -   provided that if X is O, R₃ is absent.

Preferably, the compound is a compound of formula (XIII)

In another preferred embodiment, the compound is3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione,3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione,3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione,(3R*,4S*)-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione,or(3R*,4R*)-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione.

The compounds of the invention may be administered using any amount andany route of administration effective for attenuating infectivity of thehepatitis C virus or the human immunodeficiency virus. Thus, the term“effective amount” used herein refers to a nontoxic but sufficientamount of the antiviral agent to provide the desired treatment of viralinfection. The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the infection, the particular antiviral agent and its modeof administration, and the like. The compounds are preferably formulatedin dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically discrete unit ofanti-viral agent appropriate for the patient to be treated. Each dosageshould contain the quantity of active material calculated to produce thedesired therapeutic effect either as such, or in association with theselected pharmaceutical carrier medium.

The compounds of the invention may be administered orally, parenterally,such as by intramuscular injection, intraperitoneal injection,intravenous infusion or the like, depending on the severity of theinfection being treated.

Although the compounds of the present invention can be administered toany patient who is susceptible to hepatitis C virus infection or humanimmunodeficiency virus infection, the compounds are intended for thetreatment of mammalian hosts, and especially humans.

In view of the inhibitory effect on enzyme activity produced by thecompounds of the invention, it is anticipated that these compounds willbe useful not only for therapeutic treatment of infection, but forprophylaxis, as well. The above-noted dosages will be essentially thesame whether for treatment or prophylaxis of hepatitis C virus infectionor human immunodeficiency virus.

The following examples are provided to describe the invention in furtherdetail. These examples, which set forth the best mode presentlycontemplated for carrying out the invention, are intended to illustrateand not to limit the invention.

EXAMPLE

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Example 1 General Methods for the Preparation of Compounds of thePresent Invention

Apparatus:

NMR spectra were obtained on a Varian Unity Plus 500 ¹H, 500 MHz; ¹³C,125 MHz) spectrometer. MS spectrum was measured on an electrosprayionization mass spectrometer (ESI-MS, Esquire 3000^(Plus), BrukerDaltonik GmbH, Bremen, Germany).

(1) General method for the preparation of derivatives A1˜A9, KA, KB andKC

To 0.01 mol of a compound of formula (IV) in dehydrated DMF (2 mL),0.011 mol of the corresponding amino acid was added and the mixture wasrefluxed for 10 min. at 140° C. The reaction was stopped by adding 5 mLwater and extracted with ethyl acetate (3×5 mL). The pooled ethylacetate fractions were washed with brine (1×5 mL), dried over MgSO₄ andevaporated under vacuum. The residue was purified using silica gelcolumn CHCl₃:MeOH 9.5/0.5 v/v.

2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)-4-methylpentanoicacid. (A1)

Obtained as yellow oil, ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.8 Hz,6H), 0.94 (d, J=6.4 Hz, 6H), 1.75 (s, 3H), 1.80 (s, 3H), 1.90 (m, 1H),2.06 (m, 1H), 2.3 (m, 1H), 2.52 (d, J=7.2 Hz, 2H), 4.80 (dd, J=4, 11.6Hz, 1H), 4.55 (d, J=7.2 Hz, 2H), 5.49 (m, 1H), 6.97 (d, J=8.4 Hz, 2H),7.53 (d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.1, 20.9, 22.6,22.6, 23.1, 25.2, 25.8, 28.0, 31.6, 32.8, 36.7, 37.1, 50.7, 64.8, 114.6,119.2, 121.3, 130.9, 137.5, 138.1, 138.5, 159.8, 163.2, 170.8, 171.6,173.4. ESI-MS m/z 428 [M+H].

2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)-3-phenylpropanoicacid (A2)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.78 (d, J=6.4 Hz,6H), 0.76 (d, J=6.4 Hz, 6H), 1.76 (s, 3H), 1.80 (s, 3H), 1.9 (m, 1H),2.40 (d, J=7.2 Hz, 2H), 3.50 (m, 2H), 4.52 (d, J=6.8 Hz, 2H), 5.06 (t.J=7.2, 16.4, 1H), 5.49 (m, 1H), 6.93 (d, J=8.4 Hz, 2H), 7.15 (m, 3H),7.22 (d, J=6.8 Hz, 2H), 7.39 (d, J=8.4 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz)δ18.2, 22.5, 22.6, 25.8, 27.9, 32.7, 34.4, 53.0, 64.8, 114.7, 119.2,121.1, 126.8, 128.5, 128.9, 130.8, 136.6, 137.5, 138.0, 138.6, 159.9,170.3, 171.1, 174.0. ESI-MS m/462 [M+H]⁺.

3-Hydroxy-2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicacid (A3)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.4 Hz,6H), 1.75 (s, 3H), 1.81 (s, 3H), 2.04 (m, 1H), 2.54 (d, J=8 Hz, 2H),4.12 (dd, J=3.6, 8.4 Hz, 1H), 4.23 (dd, J=5.2, 12 Hz, 1H), 4.56 (d,J=6.8 Hz, 2H), 4.90 (m, 1H), 5.49 (t, J=6.8, 13.6 Hz, 1H), 6.96 (d,J=8.6 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ18.2,22.7, 25.8, 27.9, 28.1, 32.9, 33.6, 54.9, 61.1, 64.8, 64.9, 114.8,115.1, 118.8, 119.1, 120.9, 130.9, 131.1, 138.2, 138.6, 138.7, 160.0,171.0, 171.3, 172.1. ESI-MS m/z 402 [M+H]⁺.

3-(4-Hydroxyphenyl)-2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)-phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicacid (A4)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ δ 0.78 (d, J=6.8 Hz,3H), 0.80 (d, J=6.8 Hz, 3H), 1.75 (s, 1H), 1.80 (s, 1H), 1.92 (m, 1H),2.41 (d, J=7.2 Hz, 2H), 3.43 (d, J=12.8 Hz, 2H), 4.53 (d, J=6.8 Hz, 2H),5.00 (t, J=8.4, 17.2 Hz, 1H), 5.48 (t, J=6.8, 13.6 Hz, 1H), 6.67 (d,J=8.4 Hz, 2H), 6.94 (d, J=9.2 Hz, 2H), 7.00 (d, J=9.2 Hz, 2H), 7.40 (d,J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 22.5, 22.6, 25.8, 28.0,32.7, 33.6, 53.1, 64.8, 71.1, 114.7, 115.4, 119.2, 121.1, 128.6, 130.1,130.9, 137.6, 138.1, 138.7, 154.4, 159.9, 170.4, 171.2, 173.7. ESI-MSm/z 478 [M+H]⁺.

2-(3-Isobutyl-4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-(methylthio)butanoicacid (A5)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.8 Hz,6H), 1.75 (s, 1H), 1.81 (s, 1H), 2.03 (m, 1H), 2.05 (s, 3H), 2.45 (m,4H), 2.50 (d, J=8 Hz, 2H), 4.53 (d, J=7.4 Hz, 2H), 5.00 (m, 1H), 5.49(t, J=7.2, 16.4 Hz, 1H), 6.97 (d, J=8.8 Hz, 2H), 7.53 (d, J=8.8 Hz, 2H);¹³C NMR (CDCl₃, 100 MHz) δ 15.3, 18.2, 22.7, 25.8, 27.7, 28.1, 30.9,32.9, 50.7, 64.8, 114.7, 119.1, 121.1, 130.9, 137.9, 138.3, 138.7,159.9, 170.6, 171.4, 174.7. ESI-MS m/z 446 [M+H]⁺.

3-Hydroxy-2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoicacid (A6)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.8 Hz,6H), 1.21 (d, J=6.4 Hz, 3H), 1.76 (s, 3H), 1.81 (s, 3H), 2.06 (m, 1H),2.55 (d, J=7.6 Hz, 2H), 4.55 (d, J=6.4 Hz, 2H), 4.67 (dd, J=4, 6.4 Hz,1H), 4.88 (d, J=4 Hz, 1H), 5.49 (t, J=5.2, 13.2 Hz, 1H), 6.99 (d, J=8.4Hz, 2H), 7.54 (d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 20.1,22.7, 25.8, 28.2, 32.9, 59.2, 64.9, 66.7, 114.8, 119.1, 120.8, 131.1,138.4, 138.7, 160.2, 170.5, 171.8, 172.8. ESI-MS m/z 416 [M+H]⁺.

2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methylbutanoicacid (A7)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.4 Hz,6H), 0.92 (d, J=6.4 Hz, 3H), 1.12 (d, J=6.4 Hz, 3H), 1.76 (s, 3H), 1.80(s, 3H), 2.04 (m, 1H), 2.45 (m, 1H), 2.53 (d, J=7.2 Hz, 2H), 2.67 (m,1H), 4.48 (d, J=8.0 Hz, 1H), 4.55 (d, J=−6.8 Hz, 1H), 5.48 (t, J=7.2,16.4 Hz, 1H), 6.97 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H); ¹³C NMR(CDCl₃, 100 MHz) δ 18.2, 19.4, 20.7, 22.7, 25.8, 28.1, 28.4, 32.9, 57.8,64.9, 114.8, 119.2, 121.2, 131.0, 137.6, 138.1, 138.7, 160.0, 170.8,171.6, 173.6. ESI-MS m/z 414 [M+H]⁺.

2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (A8)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.4 Hz,6H), 1.66 (d, J=7.2 Hz, 3H), 1.76 (s, 3H), 1.81 (s, 3H), 2.05 (m, 1H),2.51 (d, J=7.2 Hz, 2H), 4.55 (d, J=6.8 Hz, 2H), 4.85 (m, 1H), 5.49 (m,1H), 6.97 (d. J=8.4 Hz, 2H), 7.52 (d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100MHz) δ 15.2, 18.2, 22.7, 25.8, 28.1, 32.9, 47.4, 64.8, 114.7, 119.2,121.3, 131.0, 137.8, 138.3, 138.6, 159.9, 170.3, 171.3, 173.7; ESI-MSm/z 386 [M+H]⁺.

2-(3-Isobutyl-4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2-dihydro-1H-pyrrol-1-yl)pentanedioicacid (A9)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.88 (d, J=6.8 Hz,6H), 1.75 (s, 3H), 1.80 (s, 3H), 2.04 (m, 1H), 2.43 (br s, 4H), 2.52 (d,J=7.2 Hz, 2H), 4.55 (d, 6.8 Hz, 2H), 4.84 (dd, J=5.2, 9.2 Hz, 1H), 5.49(t, J=7.2, 16.4 Hz, 1H), 6.96 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H);¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 22.7, 23.8, 25.8, 28.1, 30.5, 32.9,50.9, 64.9, 114.8, 119.2, 121.1, 131.0, 137.9, 138.4, 138.7, 160.0,170.6, 171.4, 174.3, 177.9; ESI-MS m/z 444 [M+H].

2-Hydroxy-3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoicacid (KA)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.87 (d, J=7.2 Hz,6H), 1.75 (s, 3H), 1.81 (s, 3H), 1.99 (m, 1H), 2.51 (d, J=7.2 Hz, 2H),3.82 (m, 2H), 4.39 (dd, J=6, 7.2 Hz, 1H), 5.49 (t, J=7.2, 16.4 Hz, 1H),6.98 (d. J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz)δ 18.2, 22.8, 25.9, 28.2, 32.9, 33.9, 64.9, 114.8, 119.1, 121.0, 130.9,138.0, 138.2, 138.8, 145.1, 151.1, 160.1, 170.6, 171.9, 172.7; ESI-MSm/z 402 [M+H]⁺.

2-Hydroxy-4-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoicacid (KB)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J-6.4 Hz,6H), 1.75 (s, 3H), 1.81 (s, 3H), 2.02 (m, 1H), 2.20 (br s, 1H), 2.51 (d,J=7.6 Hz, 2H), 3.75 (m, 2H), 4.18 (br s, 1H), 4.55 (d, J=6.8 Hz, 2H),5.49 (t, J=7.2, 16.4 Hz, 1H), 6.98 (d, J=8.8 Hz, 2H), 7.49 (d, J=−8.8Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 22.8, 25.9, 28.2, 32.9, 33.9,64.9, 114.8, 119.1, 121.0, 130.9, 138.0, 138.2, 138.8, 145.1, 151.1,160.1, 170.6, 171.9, 172.7: ESI-MS m/z 416 [M+H]⁺.

2-Hydroxy-3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanoicacid (KC)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.88 (d, J=6.8 Hz,6H), 1.75 (s, 3H), 1.81 (s, 3H), 2.03 (m, 1H), 2.53 (d, J=7.2 Hz, 2H),4.55 (d, 6.8 Hz, 2H), 4.98 (d, J=4.8 Hz, 1H), 5.49 (t, J=6.4, 15.2 Hz,1H), 5.85 (d, J=4.8 Hz, 1H), 6.97 (d, J=8.4 Hz, 2H), 7.32 (m, 5H), 7.51(d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 22.7, 25.8, 28.2,32.9, 57.0, 64.9, 114.8, 115.1, 119.1, 120.7, 127.4, 128.3, 128.9,131.1, 135.5, 138.1, 138.4, 138.8, 160.3, 171.7, 172.9, 173.1; ESI-MSm/z 478 [M+H]⁺.

(2) General Method for the Preparation of Compounds A1-1˜A1-6 andA2-1˜A2˜5

A mixture of respective carboxylic acid derivative (A1-A9) (31 mmol),amino acid ester derivative (34.9 mmol) and L-hydroxybenzotriazolehydrate (38.5 mmol) were dissolved in dimethylformamide (60 mL). Theresulting solution was then placed in a water bath at 0° C. and treatedwith triethylamine (14 mL), followed by stirring for 10 min. To theresulting mixture 1-(3-(dimethyl-amino)propyl)-3-ethylcarbodiimidehydrochloride (38.5 mmol) was added. After removing the water bath, themixture was stirred for 18 h at room temperature. The reaction wasdiluted in water (200 mL), extracted with ethyl acetate, dried oversodium sulfate anhydrous, concentrated under reduced pressure andpurified by silica gel column chromatography, eluting by hexane:ethylacetate 9:1 v/v.

Methyl2-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methylpentanamido)-3-methyl-butanoate(A1-1)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.87 (d. J=6.4 Hz,6H), 0.94 (d, J=6.4 Hz, 6H), 1.45 (m, 1H), 1.76 (s, 3H), 1.81 (s, 3H),1.87 (m, 1H), 2.06 (m, 1H), 2.17 (m, 1H), 2.90 (m, 1H), 2.54 (d, J=7.2Hz, 2H), 3.72 (s, 3H), 4.54 (d, J=8 Hz, 1H), 4.55 (d. J=6.8 Hz, 2H),4.80 (dd, J=4.8, 10.8 Hz, 1H), 5.49 (m, 1H), 6.98 (d, J=8.8 Hz, 2H),7.53 (d, J=8.8 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 17.7, 18.2, 18.9,21.3, 22.7, 23.0, 25.3, 25.8, 28.1, 31.3, 32.8, 37.6, 52.1, 53.6, 57.2,64.8, 114.8, 119.1, 121.1, 130.9, 137.8, 138.2, 138.7, 160.0, 169.6,171.1, 172.0, 172.1. ESI-MS m/z 541 [M+H]⁺.

Ethyl3-(4-hydroxyphenyl)-2-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-1-yl)-4-methylpentanamido)propanoate(A1-2)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.88 (d, J=6.8 Hz,9H), 0.9 (d, J=6.8 Hz, 3H), 1.28 (t, J=7.2, 14.4 Hz, 3H), 1.39 (m, 1H),1.76 (s, 3H), 1.81 (s, 3H), 2.03 (m, 1H), 2.33 (m, 1H), 2.51 (d, J=8 Hz,2H), 2.98 (dd, J=6.4, 14.4 Hz, 1H), 3.07 (dd, 6.6, 14 Hz, 1H), 4.15 (m,2H), 4.55 (d, J=6.4 Hz, 2H), 4.75 (m, 2H), 5.49 (m, 1H), 6.66 (d, J=8.8Hz, 2H), 6.93 (d, J=8.8 Hz, 2H), 6.98 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 14.1, 18.2, 21.4, 22.7, 22.9, 25.3,25.8, 28.1, 32.8, 37.1, 37.3, 53.3, 53.6, 61.6, 64.9, 114.8, 115.4,119.1, 121.1, 127.3, 130.4, 131.0, 137.7, 138.2, 138.7, 154.9, 160.0,169.4, 171.1, 171.4, 171.9. ESI-MS m/z 619 [M+H]⁺.

Methyl3-hydroxy-2-(2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)-phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methylpentanamido)-propanoate(A1-3)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.8 Hz,6H), 0.93 (d, J=6.8 Hz, 6H) 1.45 (m, 1H), 1.76 (s, 3H), 1.81 (s, 3H),1.88 (m, 1H), 2.04 (m, 1H), 2.24 (m, 1H), 2.54 (d. J=7.2 Hz, 2H), 3.77(s, 3H), 3.98 (m, 2H), 4.55 (d, J=6.8 Hz, 2H), 4.64 (m, 1H), 4.82 (dd,J=4.4, 11.2 Hz, 1H), 5.49 (m, 1H), 6.98 (d, J=8.8 Hz, 2H), 7.53 (d,J=8.8 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 21.2, 22.7, 23.2, 25.3,25.8, 28.1, 32.8, 37.6, 52.8, 55.1, 62.8, 64.9, 114.8, 119.1, 121.0,131.0, 137.9, 138.3, 138.7, 160.1, 169.7, 170.6, 171.2, 172.1. ESI-MSm/z 529 [M+H]⁺.

Methyl1-(2-(3-Isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methylpentanoyl)pyrrolidine-2-carboxylate(A1-4)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=−6.4 Hz,6H), 0.93 (d, J=6.4 Hz, 6H), 1.27 (t, J=7.2, 14 Hz, 3H), 1.41 (d, J=6.8Hz, 3H), 1.44 (m, 1H), 1.76 (s, 3H), 1.79 (m, 1H), 1.81 (s, 3H), 2.04(m, 1H), 2.30 (m, 1H), 2.53 (d, J=7.2 Hz, 2H), 4.16 (q, 2H), 4.53 (m,1H), 4.55 (dd, J=7.2, 13.6 Hz, 3H), 4.77 (dd, J=5.2, 11.6 Hz, 1H), 5.49(m, 1H), 6.98 (d, J=−8.8 Hz, 2H), 7.53 (d, J=8.8 Hz, 2H); ¹³C NMR(CDCl₃, 100 MHz) δ 14.1, 18.2, 18.4, 21.3, 22.7, 23.1, 25.4, 25.8, 28.0,32.9, 37.6, 48.4, 53.3, 61.5, 64.9, 114.8, 119.2, 1212, 131.0, 137.7,138.2, 138.7, 160.0, 169.1, 171.2, 172.0, 172.7. ESI-MS m/z 539 [M+H]⁺.

Ethyl2-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methylpentanamido)propanoate(A1-5)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.89 (d, J=6.4 Hz,6H), 0.96 (d, J=6.4 Hz, 6H), 1.28 (t, J=14 Hz, 3H), 1.46 (d, J=7.2 Hz,3H) 1.56 (m, 1H), 1.71 (br s, 1H), 1.72 (s, 3H), 1.81 (s, 3H), 1.98 (m,2H), 2.06 (m, 2H), 2.53 (d, J=−7.2 Hz, 2H), 2.58 (m, 1H), 3.57 (m, 2H),4.50 (dd, J=−7.2, 13.6 Hz, 1H), 4.55 (d, 6.8 Hz, 2H), 4.91 (dd, J=5.2,11.6 Hz, 1H), 5.49 (m, 1H), 6.96 (d, J=8.4 Hz, 2H), 7.51 (d, J=8.4 Hz,2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 21.3, 22.7, 22.7, 23.2, 25.1,25.2, 25.8, 28.1, 28.8, 32.9, 36.4, 46.8, 51.6, 52.3, 64.8, 114.7,119.2, 121.3, 131.0, 137.5, 138.0, 138.6, 159.9, 168.6, 171.1, 171.8,172.4. ESI-MS m/z 527 [M+H]⁺.

N-Cyclopropyl-2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methylpentanamide(A1-6)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.49 (br s, 2H) 0.75(d, J=6.8 Hz, 2H), 0.90 (d, J=6.4 Hz, 6H), 0.91 (d, J=6.4 Hz, 6H), 1.71(m, 1H), 1.76 (s, 3H), 1.81 (s, 3H), 2.02 (m, 1H), 2.22 (m, 1H), 2.53(d, J=7.2 Hz, 2H), 2.71 (m, 1H), 4.55 (d, J=6.8 Hz, 2H), 4.72 (dd,J=5.2, 7.6 Hz, 1H), 5.49 (m, 1H), 6.97 (d, J=8.4 Hz, 2H), 7.52 (d, J=8.4Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 6.6, 18.3, 21.3, 22.7, 23.1, 25.4,25.8, 28.2, 32.9, 37.7, 53.7, 64.9, 114.8, 119.1, 121.3, 131.0, 137.7,138.2, 138.7, 160.1, 171.0, 171.2, 172.1. ESI-MS m/z 467 [M+H]⁺.

Methyl2-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanamido)-4-methylpentanoate(A2-1)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) (mixture of isomers) δ0.75 (d, J=6.8 Hz, 3H), 0.77 (d, J=6.8 Hz, 3H), 0.94 (d, J=6 Hz, 6H),1.54 (m, 1H), 1.64 (m, 1H), 1.75 (s, 3H), 1.77 (m, 1H), 1.81 (s, 3H),1.87 (m, 1H), 2.39 (d, J=7.2 Hz, 2H), 3.47 (m, 2H), 3.71 (s, 3H), 4.53(d, J=−6.4 Hz, 2H), 4.65 (m, 1H), 5.02 (dd, J=6.4, 10.4 Hz, 1H), 5.48(t, J=7.6, 16.7 Hz, 1H), 6.94 (d, J=8.8 Hz, 2H), 7.23 (d, J=8 Hz, 2H),7.34 (d, J=8 Hz, 2H), 7.36 (d, J=8.8 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ18.2, 21.9, 22.5, 22.7, 22.8, 24.8, 25.8, 28.0, 32.7, 34.9, 41.5, 50.9,52.3, 55.9, 64.8, 114.7, 119.1, 120.9, 126.9, 128.5, 128.9, 130.8,136.6, 137.8, 138.2, 138.7, 159.9, 168.6, 170.9, 171.7, 173.1; ESI-MSm/z 589 [M+H]⁺.

Ethyl2-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanamido)-3-phenylpropanoate.(A2-2)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) (mixture of isomers) δ0.76 (d, J=6.4 Hz, 3H), 0.74 (d, J=6.4 Hz, 3H), 1.22 (t, J=14 Hz, 3H),1.76 (s, 3H), 1.81 (s, 3H), 1.87 (m, 1H), 2.38 (d, J=7.2 Hz, 2H), 3.18(m, 2H), 3.40 (m, 2H), 4.14 (q, 2H), 4.54 (d, J 6.8 Hz, 2H), 4.85 (q,1H), 4.95 (dd, J=6.8, 10.4 Hz, 1H), 5.49 (t, J=7.2, 16.7 Hz, 1H), 6.94(d, J=8.8 Hz, 2H), 7.09 (d, J=8.4 Hz, 2H), 7.19 (m, 3H), 7.34 (d, J=8.8Hz, 2H), 7.36 (d, J=8.8 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 14.1, 18.2,22.5, 22.6, 25.8, 27.9, 32.7, 34.5, 37.9, 53.5, 55.8, 61.5, 64.8, 114.7,119.1, 120.9, 126.8, 12.9, 128.4, 128.5, 128.9, 129.3, 130.8, 135.6,136.6, 137.6, 138.1, 138.7, 159.9, 168.3, 170.8, 171.1, 171.5; ESI-MSm/z 637 [M+H]⁺.

Ethyl3-(4-hydroxyphenyl)-2-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanamido)propanoate(A2-3)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) (mixture of isomers) δ0.72 (d, J=6.8 Hz, 3H), 0.74 (d, J=6.8 Hz, 3H), 1.23 (t, J=14 Hz, 3H),1.75 (s, 3H), 1.80 (s, 3H), 1.85 (m, 1H), 2.37 (d, J=7.2 Hz, 2H), 2.90(dd, J=6.4, 11.0 Hz, 1H), 3.08 (dd, J=5.6, 14 Hz, 1H), 3.40 (m, 2H),4.14 (q, 2H), 4.54 (d, J=6.4 Hz, 2H), 4.82 (q, 1H), 4.95 (dd. J=6.4,10.8 Hz, 1H), 5.49 (t, J=7.2, 16.7 Hz, 1H), 6.65 (d, J=8.8 Hz, 2H), 6.93(d, J=8 Hz, 2H), 6.95 (d, J-8 Hz, 2H), 7.15 (m, 3H), 7.36 (d, J=8.4 Hz,2H), 7.36 (d, J=Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 14.1, 18.2, 22.5,22.6, 25.8, 27.9, 32.7, 34.5, 37.1, 53.6, 55.8, 61.6, 64.9, 114.7,115.4, 119.1, 120.9, 126.9, 127.4, 128.5, 128.9, 130.5, 130.9, 136.5,137.6, 138.1, 138.7, 154.8, 159.9, 168.5, 170.8, 171.2, 171.5; ESI-MSm/z 653 [M+H]⁺.

Methyl1-(2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanoyl)pyrrolidine-2-carboxylate(A2-4)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.78 (d, J=6.4 Hz,3H), 0.82 (d, J=6.4 Hz, 6H), 1.75 (s, 3H), 1.80 (s, 3H), 1.91 (m, 2H),2.06 (m, 2H), 2.41 (d, J=7.2 Hz, 2H), 3.37 (m, 2H), 3.58 (m, 2H), 3.71(s, 3H), 4.50 (dd, J=Hz, 1H), 4.54 (d, J=6.4 Hz, 2H), 5.07 (dd, J=Hz,1H), 5.48 (m, 1H), 6.92 (d, J=8.4 Hz, 2H), 7.12-7.24 (m, 5H), 7.39 (d,J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 18.2, 22.5, 22.7, 24.9, 25.8,27.9, 28.7, 32.7, 34.3, 46.9, 52.3, 53.9, 59.4, 64.8, 114.7, 119.2,121.1, 126.7, 128.7, 129.3, 130.9, 137.2, 137.3, 137.8, 138.7, 159.9,167.4, 170.5, 171.2, 172.6. ESI-MS m/z 573 [M+H].

N-Cyclopropyl-2-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanamide(A2-5)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.49 (br s, 2H), 0.72(d, J=6.8 Hz, 2H), 0.76 (d, J=6.8 Hz, 12H), 1.75 (s, 3H), 1.81 (s, 3H),1.85 (m, 2H), 2.66 (d, J=7.6 Hz, 2H), 2.72 (m, 1H), 3.45 (m, 2H), 4.53(d, J=7.2 Hz, 2H), 4.94 (dd, J=5.2, 10.8 Hz, 1H), 5.49 (m, 1H), 6.94 (d,J=8.4 Hz, 2H), 7.14 (m, 3H), 7.20 (d, J=8 Hz, 2H), 7.35 (d, J=8.4 Hz,2H); ¹³C NMR (CDCl₃, 100 MHz) δ 6.5, 6.6, 18.2, 22.5, 22.6, 22.8, 25.8,27.9, 32.7, 34.9, 56.3, 64.8, 114.8, 119.1, 120.9, 126.9, 128.6, 128.9,130.8, 136.6, 137.7, 138.1, 138.7, 159.9, 170.2, 171.1, 171.7. ESI-MSm/z 501[M+H]⁺.

(3) General Method for the Preparation of Compounds KA-1˜KA-4, KB-1 andKC-1˜KC-3

First step, the same method as in the preparation of compound A1-1 wasused to obtain compounds KA-1˜KA-4, KB-1 and KC-1˜KC-3. Withoutpurification, to the product from the previous step (1.6 mmol) inanhydrous dichloromethane (70 mL) was added Dess-Martin periodinane (2.4mmol) and the mixture was stirred for 18 hrs. The reaction was stoppedby adding 10% sodium thiosulfate (35 mL) and saturated sodiumbicarbonate solution (35 mL) and stirred for 30 min. the chloroformlayer was separated and washed with brine (1×10 mL) and evaporated invacuum. The resulting compound was purified on silica gel column usingHexane:ethyl acetate 9.5:0.5 v/v.

Methyl2-(3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopropanamido)-3-methylbutanoate(KA-1)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) 0.90 (d, J=6.4 Hz, 6H),0.96 (d, J=6.4 Hz, 6H), 1.76 (s, 3H), 1.81 (s, 3H), 2.07 (m, 1H), 2.24(m, 1H), 2.55 (d, J=7.2 Hz, 2H), 3.8 (s, 3H), 4.50 (dd, J=4.8, 8.8 Hz,1H), 4.55 (d, J=6.8 Hz, 1H), 4.96 (s, 2H), 5.49 (t, J=7.2, 16 Hz, 1H),6.98 (d, J=9.2 Hz, 2H), 7.53 (d, J=8.8 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz)δ 17.7, 18.2, 18.9, 22.7, 25.8, 28.1, 31.4, 32.9, 43.9, 52.4, 57.2,64.9, 114.8, 119.2, 121.2, 130.9, 138.2, 138.6, 138.7, 158.5, 160.0,170.5, 170.9, 171.3, 190.3. ESI-MS m/z 513 [M+H]⁺.

Ethyl2-(3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopropanamido)-3-phenylpropanoate(KA-2)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.90 (d, J=6.4 Hz,6H), 1.23 (t, J=6.8, 14 Hz, 3H), 1.76 (s, 3H), 1.81 (s, 3H), 2.06 (m,1H), 2.54 (d, J=7.2 Hz, 2H), 3.15 (d, J=6 Hz, 2H), 4.18 (m, 2H), 4.55(d, J=6.4 Hz, 1H), 4.82 (m, 1H), 4.92 (s, 2H), 5.49 (m, 1H), 6.98 (d,J=8.8 Hz, 2H), 7.12 (d, J=8 Hz, 2H), 7.26 (m, 3H), 7.53 (d, J=8.8 Hz,2H); ¹³C NMR (CDCl₃, 100 MHz) δ 14.1, 18.3, 22.7, 25.8, 28.1, 32.9,38.0, 43.8, 53.2, 61.9, 64.9, 114.8, 119.2, 121.2, 127.4, 128.7, 129.2,130.9, 135.0, 138.3, 138.6, 138.7, 158.2, 160.0, 170.1, 170.5, 171.2,190.1. ESI-MS m/z 575 [M+H]⁺.

Ethyl2-(3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopropanamido)propanoate(KA-3)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.91 (d, J=6.8 Hz,6H), 1.28 (t, J=7.2, 14 Hz, 3H), 1.46 (d, J=7.2 Hz, 3H), 1.76 (s, 3H),1.81 (s, 3H), 2.07 (m, 1H), 2.54 (d, J=7.6 Hz, 2H), 4.23 (q, 2H), 4.56(d, J=6.4 Hz, 2H), 4.92 (s, 2H), 5.49 (t, J=7.2, 16.7 Hz, 1H), 6.99 (d,J=8.4 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 14.1,18.1, 18.3, 22.7, 25.8, 28.1, 32.9, 43.8, 48.1, 61.9, 64.9, 114.8,119.2, 121.2, 130.9, 138.2, 138.6, 138.7, 158.1, 160.0, 170.5, 171.3,171.5, 190.3. ESI-MS m/z 499 [M+H]⁺.

N-Cyclopropyl-3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxopropanamide(KA-4)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.62 (br s, 2H), 0.85(d, J=5.6 Hz, 2H), 0.91 (d, J=6.4 Hz, 6H), 1.76 (s, 3H), 1.81 (s, 3H),2.07 (m, 1H), 2.55 (d, J=7.2 Hz, 2H), 2.78 (m, 1H), 4.55 (d, J=6.8 Hz,2H), 4.96 (s, 2H), 5.49 (t, J=7.2, 16 Hz, 1H), 6.98 (d, J=8.8 Hz, 2H),7.52 (d, J=8.8 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 6.5, 18.2, 22.4,22.7, 25.8, 28.1, 32.9, 43.8, 64.9, 114.8, 119.2, 121.2, 130.9, 138.2,138.5, 138.7, 160.0, 160.1, 171.3, 190.9. ESI-MS m/z 439 [M+H]⁺.

N-Cyclopropyl-4-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxobutanamide(KB-1)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.59 (br s, 2H), 0.83(d, J=5.6 Hz, 2H), 0.89 (d, J=6.4 Hz, 6H), 1.76 (s, 3H), 1.81 (s, 3H),2.03 (m, 1H), 2.49 (d, J=7.6 Hz, 2H), 2.76 (m, 1H), 3.2 (t, J=6.8, 13.6Hz, 2H), 3.9 (t, J=6.8, 13.6 Hz, 2H), 4.55 (d, J=6.8 Hz, 2H), 5.49 (t,J=7.2, 16 Hz, 1H), 6.97 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H); ¹³CNMR (CDCl₃, 100 MHz) δ 6.5, 18.2, 22.4, 22.7, 25.8, 28.1, 32.8, 32.9,35.8, 64.8, 114.7, 119.2, 121.3, 130.9, 137.9, 138.2, 138.7, 159.9,160.9, 171.0, 171.7, 196.6. ESI-MS m/z 453 [M+H]⁺.

Ethyl2-(3-(3-isobutyl-4-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-3-phenylpropanamido)-3-phenylpropanoate(KC-1)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.88 (d, J=6.4 Hz,3H), 0.91 (d, J=6.4 Hz, 3H), 1.20 (t, J=7.2, 14 Hz, 3H), 1.76 (s, 3H),1.80 (s, 3H), 2.04 (m, 1H), 2.51 (d, J=7.2 Hz, 2H), 3.11 (m, 2H), 4.14(q, J=Hz, 2H), 4.53 (d, J=6.8 Hz, 2H), 4.72 (m, 1H), 5.49 (t, J=7.2, 16Hz, 1H), 6.30 (s, 1H), 6.35 (s, 1H), 6.95 (d, J=8.8 Hz, 2H), 7.28 (br s,5H), 7.36 (br s, 5H), 7.51 (d, J=8.8 Hz, 2H); ESI-MS m/z 651 [M+H]⁺.

Ethyl2-(3-(3-isobutyl-(4-(3-methylbut-2-enyloxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-3-phenylpropanamido)propanoate(KC-2)

Obtained as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ 0.88 (d, J=6.4 Hz,6H), 1.25 (t, J=7.2, 14 Hz, 3H), 1.41 (d, J=6.8 Hz, 3H), 1.76 (s, 3H),1.80 (s, 3H), 2.04 (m, 1H), 2.51 (d, J=7.2 Hz, 2H), 4.19 (m, 2H), 4.44(m, 1H), 4.53 (d, J=6.8 Hz, 2H), 5.49 (t, J=7.2, 16 Hz, 1H), 6.3 (s,1H), 6.32 (s, 1H), 6.95 (d, J=8.8 Hz, 2H), 7.36 (br s, 5H), 7.51 (d,J=8.4 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 14.1, 17.9, 17.9, 18.2, 22.7,25.8, 28.1, 32.9, 48.2, 48.3, 58.6, 61.7, 61.8, 64.9, 114.8, 119.2,121.1, 121.2, 128.7, 128.9, 129.2, 129.3, 130.9, 131.0, 133.7, 138.1,138.4, 138.5, 138.7, 158.9, 159.1, 159.9, 171.3, 171.5, 191.4. ESI-MSm/z 575 [M+H]⁺.

(4) Compounds A1˜A9, A1-1˜A1-5 and A2-1˜A2-4 were Prepared According tothe Process Outlined in Scheme 1 as Shown Below.

Reaction conditions: (a) amino acid, DMF, 1400° C.; (b) Amino acidester, DMF, HOBT, triethylamine, 0° C., EDC, rt 18 hrs.

Representative compounds of Scheme 1 were as listed in Tables 1-2,below.

TABLE 1 Representative Compounds of Formula (V) R₁ Compound No.—CH₂—CH—(CH₃)₂ A1 —CH₂—Ph A2 —CH₂—OH A3 —CH₂-(p-OH—Ph) A4 —(CH₂)₂—SH—CH₃A5 —CH(OH)—CH₃ A6 —CH—(CH₃)₂ A7 —CH₃ A8 —(CH₂)₂—COOH A9

TABLE 2 Representative Compounds of Formula (VI) R₁ R₂ R₃ Compound No.—CH₂—CH—(CH₃)₂ —CH—(CH₃)₂ Me A1-1 —CH₂—CH—(CH₃)₂ —CH₂-(p-OH—Ph) EthylA1-2 —CH₂—CH—(CH₃)₂ —CH₂—OH Me A1-3 —CH₂—CH—(CH₃)₂ Proline Me A1-4—CH₂—CH—(CH₃)₂ —CH₃ Ethyl A1-5 —CH₂—Ph —CH₂—CH—(CH₃)₂ Me A2-1 —CH₂—Ph—CH₂—Ph Ethyl A2-2 —CH₂—Ph —CH₂—P—OHPh Ethyl A2-3 —CH₂—Ph Proline MeA2-4(5) Compounds KA˜KC, KA-1˜KA-3 and KC-1˜KC-2 were Prepared According tothe Process Outlined in Scheme 2 as Shown Below.

Reaction conditions: (a) amino acid, DMF, 140° C.; (b) Amino acid ester,DMF, HOBT, tri-ethylamine, 0° C., EDC, rt 18 hrs; (c) Dess-martinperiodinane, CH₂Cl₂, rt, 18 hrs.

Representative compounds of Scheme 2 were as listed in Tables 3-5,below.

TABLE 3 Representative Compounds of Formula (VII) n R₁ Compound No. 1 —HKA 2 —H KB 1 Phenyl KC

TABLE 4 Representative Compounds of Formula (VIII) n R₁ R₂ R₃ CompoundNo. 1 —H —CH—(CH₃)₂ Me None 1 —H —CH₂—Ph Ethyl None 1 —H —CH₃ Ethyl None1 Phenyl —CH₂—Ph Ethyl None 1 Phenyl —CH₃ Ethyl None

TABLE 5 Representative Compounds of Formula (IX) n R₁ R₂ R₃ Compound No.1 —H —CH—(CH₃)₂ Me KA-1 1 —H —CH₂—Ph Ethyl KA-2 1 —H —CH₃ Ethyl KA-3 1Phenyl —CH₂—Ph Ethyl KC-1 1 Phenyl —CH₃ Ethyl KC-2(6) Compounds KB-1, KC-3, A1-6 and A2-5 were Prepared According to theProcess Outlined in Scheme 3 as Shown Below.

Reaction conditions: (b) Amino acid ester, DMF, HOBT, tri-ethylamine, 0°C., EDC, rt 18 has; (c) Dess-martin periodinane, CH₂Cl₂, rt, 18 hrs.

Representative compounds of Scheme 3 were as listed in Tables 6-8,below.

TABLE 6 Representative Compounds of Formula (X) n R₁ Compound No. 1Phenyl None 2 —H None 1 —CH₂—CH—(CH₃)₂ None 1 —CH₂—Ph None

TABLE 7 Representative Compounds of Formula (XI) n R₁ Compound No. 1Phenyl None 2 —H None 1 —CH₂—CH—(CH₃)₂ None 1 —CH₂—Ph None

TABLE 8 Representative Compounds of Formula (XII) n R₁ Compound No. 1Phenyl KC-3 2 H KB-1 1 —CH₂—CH—(CH₃)₂ A1-6 1 —CH₂—Ph A2-5(7) The R₃ group of the compounds of formula (I) was prepared from themethod as described in reference (Stewart et al., 2007, A concisesynthesis of maleic anhydride and maleimide natural products found inAntrodia camphorate. Tetrahedron Letters, 48, 2241-2244; Cheng et al.,2008, Total synthesis of (±)-camphorataimides and (±)-himanimides byNaBH₄/Ni(OAc)₂ or Zn/AcOH stereoselective reduction. Tetrahedron, 64,4347-4353).

Example 2 HCV Protease Assay

The HCV protease assay herein was applied to investigate theHCV-protease inhibitory activity of the prepared compounds as describedabove. The method of the HCV protease assay was described in D. T.Phuong, C. M. Ma, M. Hattori and J. S. Jin: Inhibitory Effects ofAntrodins A-E from Antrodia cinnamomea and Their Metabolites onHepatitis C Virus Protease. Phytotherapy Research, 23, 582-584, 2009.Two micro liters of a compound solution (using DMSO as solvent) wasplaced in 384 well micro plate, then 8 μl of HCV NS3/4A protease (0.5g/mL) was added to the well containing a sample and the plate wasagitated. Finally, 10 μL of freshly prepared substrate(Ac-Asp-Glu-Dap(QXL™ 520)-Glu-Glu-Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH₂)(100× dilution of a DMSO stock solution) was added with sequentialrotational shaking. The reaction mixture was incubated for 30 min at 37°C. The fluorimetric analyses were performed on an automated TECAN GENiosplate reader with excitation wavelength at 485 nm and emission at 530nm. Each test compound was carried out in triplicate. The HCV-PRinhibition (%) was calculated by using the following equation:

% inhibition=(F_(vehicle) control−F_(substrate) control)×100/F_(vehicle)control

(Where F is the fluorescence value of vehicle control or of sample minusthe fluorescence of the substrate control).

The results were shown in Table 9.

In addition, the dipeptidyl peptidase-IV (DPPIV) assay herein wasapplied to investigate the DPPIV inhibitory activity of the preparedcompounds as described above. The method of the DPPIV assay wasdescribed in Lin et al., 1998. Inhibition of dipeptidyl peptidase IV byfluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics.Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14020-14024. The DPPIVinhibition (%) was calculated by using the following equation:

% inhibition=[(1−v _(i) /v ₀)]×100

(Where v_(i) and v₀ are the V_(max) value of test compound and control,respectively).

The results were shown in Table 10.

TABLE 9 IC₅₀ values of the compounds against HCV protease.

Compound No. R IC₅₀ (μM) A1

8.6 A2

14.0 A3

27.0 A4

16.0 A5

21.0 A6

18.0 A7

23.3 A8

27.0 A9

19.0 KA

8.7 KB

15.6 KC

5.0 A1-1

>100 A1-2

>100 A1-3

>100 A1-4

>100 A2-1

>100 A2-2

100 A2-3

100 A1-6-ter

>200 A1-7-ter

>200 A1-10-ter

>200 A2-11-ter

>200 KB-3

>100 KC-1

>100 KC-2

>100 KC-3

>100 KAnh-1

153 KBnh-1

12.1 KCnh-1

1.95 KA-5-2

47.6 KB-1-2

35.2 KB-2-2

16.2 KC-4-2

18.3 KC-5-2

51.6

Compound No. R IC₅₀ (μM) A10

>200 A11

63 A12

25 A13

>200 A14

>200 A15

85 A16

170 A17

105 KA-t

185 KB-t

75 KC-t

73 A1-6

>200 A1-7

27 A1-10

16 A2-11

120 A2-12

192 A3-16

132 KAnh-2

145.5 KBnh-2

7.4 KCnh-2

1.4 KA-5

70 KB-1

61.9 KB-2

100 KC-4

14.6 KC-5

9

TABLE 10 IC₅₀ values of the compounds against DPPIV

Compound No. R IC₅₀ (μg/ml) Antrodin A X═O  25 Antrodin B X═N  50Antrodin C X═N, R═OH  38 A1

 45 A2

 35 A3

 40 A1-5

>100*   35* A1-8

>100*   50* A2-6

    89.0*   70* A2-8

>100*   43* A1-9-ter

 60 A2-14-ter

 84 A3-15-ter

 55

Comp No. R IC₅₀ (μM) A1-t

75 A2-t

90 A3-t

70 A1-9

29 A2-14

60 A3-15

85 Note: 1.

denotes the place of connection. 2. Positive control (HCV proteaseinhibitor I (Anaspec) was 1.5 μM. 3. *represents that the two values arefrom independent experiments of the same compound.

According to Table 9, most of the compounds of A1 to A9 and KA to KCshowed significant inhibition of HCV protease with IC₅₀ ranging from 5μM to 27 μM. For other compounds showing less inhibitive activities,they were still useful in treating or prophylaxis of HCV infection aslong as the amount was large enough.

Example 3 New Compound (Formula (XIII)) Isolated from the Mycellium ofAntrodia cinnamomea

The EtOH extract was prepared from the mycelium of Antrodia cinnamomea(450 g). The extract was separated by silica gel column chromatography,and it was divided into Fr.1-Fr.10. Moreover, Fr.9 which containedCompound 4 was separated by silica gel column chromatography to obtainFr.12. Fr.12 was analyzed by LC-MS. The new compound, together withknown Compounds 4 and 5, were confirmed after HPLC isolation (FIGS.1-3).

The new compound was isolated as a yellow oily compound with molecularformula: C₁₉H₂₃O₄N ([M]⁺ m/329.16271, HR-EI-MS), specific rotation:[α]₂₃ ^(D) ±0° (c 0.276, CHCl₃). The UV spectrum showed the maximumwavelength of absorption at 280 nm, suggesting the presence of phenylring(s) in the molecule. Moreover, according to the IR spectralanalysis, a hydroxyl group (3019 cm⁻¹) and carbonyl group (1714 cm⁻¹)were also predicted to be present.

The structure of new compound was finally determined by ¹H-NMR, ¹³C-NMR(Table 11) and HMBC, HMQC spectra (FIG. 4).

TABLE 11 ¹H-NMR and ¹³C-NMR spectral data of a new compound (300 MHz and75 MHz, respectively) in CDCl₃ Position δ_(H) δ_(C) 1 — — 2 — 167.0 3 —150.0 4 4.38 (d, J = 2.0 Hz) 46.2 5 — 171.2 1′ 6.77 (d, J = 8.8, 2.0 Hz)148.5 2′ 2.24-2.30 (m) 28.8 3′ 0.72 (d, J = 6.6 Hz) 21.5 4′ 0.99 (d, J =6.6 Hz) 20.6 1″ — 119.3 2″ 7.15 (d, J = 8.8 Hz) 129.0 3″ 6.88 (d, J =8.8 Hz) 115.3 4″ — 159.0 5″ 6.88 (d, J = 8.8 Hz) 115.3 6″ 7.15 (d, J =8.8 Hz) 129.0 1′″ 4.48 (d, J = 6.3 Hz) 64.8 2′″ 5.44-5.47 (m) 119.3 3′″— 139.4 4′″ 1.79 (s) 18.3 5′″ 1.73 (s) 25.8

The structure of a new compound was shown as formula (XIII).

According to the HPLC analysis with a chiral column, a compound offormula (XIII) was showed two peaks with very close retention-times(FIG. 5). The MS² spectral pattern at m/z 330 in the MS (MS¹) spectrawere essentially identical with each other (FIG. 6).

On the basis of the specific optical rotation value: [α]_(D) ²³ ±0°, thecompound of formula (XIII) was confirmed as a racemic form. Thus, thecurve line indicated that the compound of formula (XIII) was a racemicmixture of (4S)- and (4R)-[4-(3-methyl-2-butenyloxy)phenyl]derivativeswith a 1:1 ratio. In addition, appreciable Nuclear Overhauser Effect(NOE) was found between H-1′ and H-4, showing that an exo-cyclic doublebond is located at C-1′ and C-3 with a Z-type.

A new compound of formula (XIII) with a double bond in the side chainwas isolated from the mycelium of Antrodia cinnamomea by extracting withEtOH, and the structure was determined by spectroscopic means. Forverification, powder of mycelium of Antrodia cinnamomea was extractedwith a different organic solvent CHCl₃ and analyzed by LC-MS in the sameway. The same peak was confirmed (data not shown). These findings showedthat the compound of formula (XIII) was able to be extracted with bothsolvents, there was no big difference between CHCl₃ and EtOHextractions.

Example 4 NS3/4A HCV Protease Inhibition Assay for DeterminingInhibitive Activity of a Compound of Formula (XIII)

When sensitive cells were infected with HCV virus, some precursorproteins were translated from uncoated virus RNA in the cytoplasm.According to the protease, the precursor proteins were processed intodifferent viral proteins. Among viral proteins, NS3, a serine proteaseand RNA helicase, was an important non-structural protein involved invirus protein maturation and virus genome replication.

In this assay, protein (FRET) was prepared and modified with fluorescentprotein 5-FAM as a substrate and QXL™ 520 as a quencher. The substratewas incubated with NS3/4A HCV protease and testing samples, and thefluorescence intensity was measured, according to the mechanism shown inFIG. 7 (C. M. Ma, Y. Wei, Z. G. Wang and M. Hattori: Triterpenes fromCynomorium Smngaricium-analysis of HCV Protease Inhibitory Activity,Quantification and Content Change Under the Influence of Heating. J.Nat. Med., 63, 9-14, 2009). In this assay, HCV NS3 Protease inhibitor 2was used as positive control.

Result:

For the compound of formula (XIII), a 50% NS3/4A HCV protease inhibitoryconcentration (IC₅₀) was shown in Table 12.

TABLE 12 NS3/4A HCV protease inhibitive activity of a compound offormula (XIII) Sample Structure IC₅₀ (μM) Antrodin F

— 8.7 Positive control HCV NS3 Protease Inhibitor 2 — 1.9

Example 5 HIV-1 Protease Inhibition Assay for Determining InhibitiveActivity of a Compound of Formula (XIII) and Compounds 1-5

HIV was a retrovirus with a single-stranded RNA genome. HIV protease wasan important enzyme for replication. Translated proteins in the hostcell were cut into the desired shape by the enzyme. The substance was aprotein dimer consisting of 99 amino acids, and the active center wasAsp residues.

In this assay, oligopeptide(His-Lys-Ala-Arg-Val-Leu-Phe(NO2)-Glu-Ala-NLe-Ser-NH2) with strengthenedabsorbance was prepared as a substrate, in which para site of Aspresidue was modified by nitro. The substrate was incubated in thepresence of HIV-1 protease and the test samples. Generation ratio ofhydrolysis product (Phe (NO2)-Glu-Ala-NLe-Ser-NH2) was analyzed by HPLC,and the HIV-1 inhibitory activity in test sample was detected (Y. Wei,C. M. Ma, D. Y. Chen and M. Hattori: Anti-HIV-1 Protease Triterpenoidsfrom Stauntonia Obovatifoloala Hayata Subsp. Phytochemistry, 69,1875-1879, 2008). In this assay, pepsatatin A was used as a positivecontrol.

Result:

The inhibitory activity of a compound of formula (XIII) and compounds1-5 (U.S. Pat. No. 7,109,232), ingredients in the mycelium of Antrodiacinnamomea, was represented as a 50% inhibitory concentration (IC₅₀)against HIV-1 protease (Table 13).

TABLE 13 HIV-1 protease inhibitive activity of a compound of formula(XIII) and compounds 1-5 Sample Structure IC₅₀ (μM) Compound 1 Compound2 Compound 3

X═O X═NH X═N—OH >300 54 6.5 Compound 4 Compound 5

3R*, 4S* 3R*, 4R* 4.4 198.3 (Compound of formula (XIII)

— 16.6 Positive control Pepsatin — 0.5

Compound 4 showed the highest inhibitory activity, followed by compound3, which was followed by a compound of formula (XIII) and compound 2.Compound 5 was a structural isomer of compound 4, but it showeddifferent activity between them. Although activity of compound 1 waslowest, compound 1 was useful in treating or prophylaxis of HIVinfection as long as the amount was large enough.

What is claimed is:
 1. A compound of formula (I)

where R₁ is

or H; R₂ is selected from the groups as follows:

R₃ is C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, benzyl, or4-(3-methyl-2-butenyloxy)phenyl,

denotes the place of connection, or pharmaceutically acceptable salts orprodrugs thereof.
 2. The compound of claim 1, where R₁ is

R₂ is selected from the groups as follows:

R₃ is isobutyl,

denotes the place of connection.
 3. The compound of claim 1, where R₁ isH R₂ is selected from the groups as follows:

R₃ is isobutyl,

denotes the place of connection.
 4. A compound having formula (II),

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl, or pharmaceutically acceptable saltsor prodrugs thereof.
 5. The compound of claim 4, wherein the compound is


6. A method for treating or prophylaxis of hepatitis C virus (HCV)infection which comprises administering to a subject in need thereof aneffective amount of an active agent selected from (1) a compound ofclaim 1; or (2) a compound having the formula (II)

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl.
 7. The method of claim 6, whereinthe compound is


8. The method of claim 6, wherein the compound having the formula (XIV)

wherein R is

denotes the place of connection.
 9. The method of claim 8, wherein R is


10. The method of claim 6, wherein the subject is a mammal.
 11. Themethod of claim 10, wherein the mammal is a human.
 12. A method fortreating or prophylaxis of human immunodeficiency virus (HIV) infectionwhich comprises administering to a subject in need thereof an effectiveamount of an active agent selected from (1) a compound having theformula (II)

wherein R₁ is C₁₋₁₀ alkyloxy, C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; andR₂ is C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl; or (2) a compound having the formula(III)

wherein

denotes a single or double bond; X is N or O; R₁ is C₁₋₁₀ to alkyloxy,C₂₋₁₀ alkenyloxy, or C₂₋₁₀ alkynyloxy; R₂ is H, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl or C₂₋₁₀ alkynyl; and R₃ is absent, H or hydroxy; provided thatif X is O, R₃ is absent.
 13. The method of claim 12, wherein thecompound is


14. The method of claim 12, wherein the compound is3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione,3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione,3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione,(3R*,4S*)-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione,or(3R*,4S*)-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2,5-dione.15. The method of claim 12, wherein the subject is a mammal.
 16. Themethod of claim 15, wherein the mammal is a human.